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Abstract Background In non-small cell lung cancer (NSCLC), the rapid advancement of predictive genetic testing of tumors by identifying specific pathogenic driver variants has significantly improved treatment guidance. However, immune checkpoint blockade (ICB) is typically administered to patients with tumors in the absence of such driver variants. Since only about 30% of patients will respond to ICB treatment, identifying novel genetic biomarkers of clinical response is crucial and will improve treatment decisions. This prospective clinical study aims to combine molecular biology, advanced bioinformatics and clinical data on response to treatment with ICB from a prospective cohort of NSCLC patients to identify single or combination of genetic variants in the tumor that can serve as predictive biomarkers of clinical response. Methods In this prospective bi-center clinical study, we performed next-generation sequencing (NGS) of 597 cancer-associated genes in a prospective cohort of 49 patients as the final cohort analyzed, with stage III or IV NSCLC, followed by establishment of an in-house developed bioinformatics-based molecular classification method that integrates, interprets and evaluates data from multiple databases and variant prediction tools. Overall survival (OS) and progression-free survival (PFS) were analyzed for selected candidate genes and variants identified using our novel methodology including molecular tools, databases and clinical information. Results Our novel molecular interpretation and classification method identified high impact variants in frequently altered genes KRAS, LRP1B, and TP53. Analysis of these genes as single predictive biomarkers in ICB-treated patients revealed that the presence of likely pathogenic variants and variants of unclear significance in LRP1B was associated with improved OS (p = 0.041). Importantly, further analysis of variant combinations in the tumor showed that co-occurrence of KRAS and LRP1B variants significantly improved OS (p = 0.003) and merged PFS (p = 0.008). Notably, the triple combination of variants in KRAS, LRP1B, and TP53 positively impacted both OS (p = 0.026) and merged PFS (p = 0.003). Conclusions This study suggests that combination of the LRP1B and KRAS variants identified through our novel molecular classification scheme leads to better outcomes following ICB treatment in NSCLC. The addition of TP53 improves the outcome even further. To our knowledge, this is the first report indicating that harboring a combination of KRAS, LRP1B, and TP53 variants can significantly enhance the response to ICB, suggesting a novel predictive biomarker combination for NSCLC patients.

ABSTRACT Pulmonary veno‐occlusive disease (PVOD) is a lethal variant of pulmonary hypertension. The degree of pulmonary arterial involvement varies. Here, we compare two PVOD patients who were transplanted at 8 years of age, whereof one is a homozygous EIF2AK4 mutation carrier. Tissue was imaged with synchrotron‐based micro‐CT and the results were compared with clinical data and sectioned tissue was analyzed with histology, immunohistochemistry, immunofluorescence, and in situ hybridization. Chest CT of the noncarrier exhibited scattered poorly defined ground‐glass opacities and marked septal lines, whereas the mutation carrier showed numerous nodular centrilobular ground‐glass opacities and sparse septal lines. The noncarrier developed pulmonary edema with vasodilators and 3D imaging combined with histology showed severe obstruction of interlobular septal veins and medial hypertrophy of pulmonary arteries, but no arterial or arteriolar intimal fibrosis. In contrast, the mutation carrier exhibited only mild intimal fibrosis in interlobular septal veins but severe arterial and arteriolar remodeling, including intimal fibrosis, tortuous course of arterioles, muscularization extending to the alveolar duct level and multiple vascular lumens within the same pulmonary arterial adventitia. Both patients had focally thickened alveolar septa with areas of pulmonary capillary hemangiomatosis (PCH) which colocalized with increased capillary muscularization, tenascin C expression, and deposition, as well as with matrix metalloproteinase‐9 (MMP9)/CD45 positive cells. In conclusion, synchrotron‐based phase‐contrast micro‐CT is valuable for understanding vascular remodeling. Significant differences were observed between heritable and sporadic PVOD, which may influence management strategies.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15–16 and 23–24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNACT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers.

Background: In non-small cell lung cancer (NSCLC), the rapid advancement of predictive genetic testing of tumors by identifying specific pathogenic driver variants has significantly improved treatment guidance. However, immune checkpoint blockade (ICB) is typically administered to patients with tumors in the absence of such driver variants. Since only about 30% of patients will respond to ICB treatment, identifying novel genetic biomarkers of clinical response is crucial and will improve treatment decisions. This prospective clinical study aims to combine molecular biology, advanced bioinformatics and clinical data on response to treatment with ICB from a prospective cohort of NSCLC patients to identify single or combination of genetic variants in the tumor that can serve as predictive biomarkers of clinical response. Methods: In this prospective bi-center clinical study, we performed next-generation sequencing (NGS) of 597 cancer-associated genes in a prospective cohort of 49 patients as the final cohort analyzed, with stage III or IV NSCLC, followed by establishment of an in-house developed bioinformatics-based molecular classification method that integrates, interprets and evaluates data from multiple databases and variant prediction tools. Overall survival (OS) and progression-free survival (PFS) were analyzed for selected candidate genes and variants identified using our novel methodology including molecular tools, databases and clinical information. Results: Our novel molecular interpretation and classification method identified high impact variants in frequently altered genes KRAS, LRP1B, and TP53. Analysis of these genes as single predictive biomarkers in ICB-treated patients revealed that the presence of likely pathogenic variants and variants of unclear significance in LRP1B was associated with improved OS (p = 0.041). Importantly, further analysis of variant combinations in the tumor showed that co-occurrence of KRAS and LRP1B variants significantly improved OS (p = 0.003) and merged PFS (p = 0.008). Notably, the triple combination of variants in KRAS, LRP1B, and TP53 positively impacted both OS (p = 0.026) and merged PFS (p = 0.003). Conclusions: This study suggests that combination of the LRP1B and KRAS variants identified through our novel molecular classification scheme leads to better outcomes following ICB treatment in NSCLC. The addition of TP53 improves the outcome even further. To our knowledge, this is the first report indicating that harboring a combination of KRAS, LRP1B, and TP53 variants can significantly enhance the response to ICB, suggesting a novel predictive biomarker combination for NSCLC patients. [ABSTRACT FROM AUTHOR]

ObjectivesImmunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3–4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response.Materials and methodologyBlood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months’ assessment after the start of therapy.ResultsWe report a significant increase in the post-treatment frequency of activated effector memory CD4+ and CD8+ T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders.ConclusionCombined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.

Objectives Efficacy and safety of tofacitinib, an oral Janus kinase inhibitor, were evaluated in a 6-month, double-blind, phase 3 study in Chinese patients with active (polyarthritic) psoriatic arthritis (PsA) and inadequate response to ≥1 conventional synthetic disease-modifying antirheumatic drug.Methods Patients were randomised (2:1) to tofacitinib 5 mg twice daily (N=136) or placebo (N=68); switched to tofacitinib 5 mg twice daily after month (M)3 (blinded). Primary endpoint: American College of Rheumatology (ACR50) response at M3. Secondary endpoints (through M6) included: ACR20/50/70 response; change from baseline in Health Assessment Questionnaire-Disability Index (HAQ-DI); ≥75% improvement in Psoriasis Area and Severity Index (PASI75) response, and enthesitis and dactylitis resolution. Safety was assessed throughout.Results The primary endpoint was met (tofacitinib 5 mg twice daily, 38.2%; placebo, 5.9%; p

Pulmonary veno‐occlusive disease (PVOD) is a lethal variant of pulmonary hypertension. The degree of pulmonary arterial involvement varies. Here, we compare two PVOD patients who were transplanted at 8 years of age, whereof one is a homozygous EIF2AK4 mutation carrier. Tissue was imaged with synchrotron‐based micro‐CT and the results were compared with clinical data and sectioned tissue was analyzed with histology, immunohistochemistry, immunofluorescence, and in situ hybridization. Chest CT of the noncarrier exhibited scattered poorly defined ground‐glass opacities and marked septal lines, whereas the mutation carrier showed numerous nodular centrilobular ground‐glass opacities and sparse septal lines. The noncarrier developed pulmonary edema with vasodilators and 3D imaging combined with histology showed severe obstruction of interlobular septal veins and medial hypertrophy of pulmonary arteries, but no arterial or arteriolar intimal fibrosis. In contrast, the mutation carrier exhibited only mild intimal fibrosis in interlobular septal veins but severe arterial and arteriolar remodeling, including intimal fibrosis, tortuous course of arterioles, muscularization extending to the alveolar duct level and multiple vascular lumens within the same pulmonary arterial adventitia. Both patients had focally thickened alveolar septa with areas of pulmonary capillary hemangiomatosis (PCH) which colocalized with increased capillary muscularization, tenascin C expression, and deposition, as well as with matrix metalloproteinase‐9 (MMP9)/CD45 positive cells. In conclusion, synchrotron‐based phase‐contrast micro‐CT is valuable for understanding vascular remodeling. Significant differences were observed between heritable and sporadic PVOD, which may influence management strategies. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a chronic skin disease with increasing prevalence worldwide. It is characterized by pruritic eczematous lesions, affecting up to 20% of the children and negatively impacting their quality of life. Guidelines for AD management are available worldwide, but specific guidelines for pediatric AD in Saudi Arabia are lacking. This consensus document aims to identify the needs for the diagnosis and management of pediatric AD in Saudi Arabia by gathering the opinions and recommendations of key experts. We conducted a three-step modified Delphi method to develop the present consensus. The experts agreed that pediatricians and dermatologists commonly encounter AD; however, it is still under-recognized in its early stage in Saudi Arabia. The family physicians should be involved in assessing suspected children with a family history of atopy, particularly in patients with isolated lesions. Further, the experts confirmed that AD diagnosis should be documented, showing assessment criteria used, key morphological characteristics, and features used to ascertain the severity of the disease. There is still a need for simple validated diagnostic criteria suitable for daily practice for pediatric AD. The experts highlighted several medical conditions that pertain to the diagnosis and management of AD in Saudi Arabia.

Objective: Current vascular grafts all have limitations. This study examined peritoneum as a potential graft material and the in vivo transfer of peritoneum into a functional artery like conduit after end to end anastomosis into the common carotid artery of sheep. The aim was to investigate whether implantation of a peritoneal tube into the arterial tree results in a structure with function, histological findings, and gene expression like an artery, and whether such arterialisation occurs through a conversion of the phenotype of peritoneal cells or from host cell migration into the implant. Methods: Peritoneum with adherent rectus aponeurosis from sheep was used to form tubular vascular grafts that were implanted into the common carotid artery of six sheep, then removed after five months. Two sheep received allogenic peritoneal grafts and four sheep received autologous peritoneal grafts. Results: One sheep died shortly after implantation, so five of the six sheep were followed. Five months after implantation, four of the five remaining grafts were patent. Three of four patent grafts were aneurysmal. The four patent grafts had developed an endothelial layer indistinguishable from that of the adjacent normal artery, and a medial layer with smooth muscle cells with a surrounding adventitia. The new conduit displayed vasomotor function not present at the time of implantation. DNA genotyping showed that the media in the new conduit consisted of recipient smooth muscle cells. Little difference in mRNA expression was demonstrated between the post-implantation conduit and normal artery. Conclusion: During a five month implantation period in the arterial system, peritoneum converted into a tissue that histologically and functionally resembled a normal artery, with a functional genetic expression that resembled that of an artery. Single nucleotide polymorphism analysis indicated that this conversion occurs through host cell migration into the graft.

Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15–16 and 23–24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNACT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers. [ABSTRACT FROM AUTHOR]

Over 40 years ago, abnormal enlargement of the nucleus of tubular epithelial cells was reported in a rare distinct hereditary chronic interstitial nephritis, karyomegalic interstitial nephritis (KIN). Here, we report the second case of systemic karyomegaly with pulmonary manifestations and present a detailed characterization of the karyomegalic cells in lung parenchyma. A 59-year-old woman who was diagnosed with KIN developed renal failure and eventually received a renal transplant later evaluated for chronic and progressive restrictive lung disease. The KIN diagnosis prompted us to carefully examine her lung parenchyma. Karyomegalic cells were identified in the alveolar epithelium, interstitium, as well as, in the vascular wall. Viral serological and biochemical blood analyses were negative. We consider that the pulmonary manifestations of karyomegaly expands the differential diagnosis of interstitial lung disease in patients with KIN. Keywords: Karyomegaly, Interstitial lung disease, Karyomegalic interstitial nephritis

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis.

Dietary antioxidants and supplements are widely used to protect against cancer, even though it is now clear that antioxidants can promote tumor progression by helping cancer cells to overcome barriers of oxidative stress. Although recent studies have, in great detail, explored the role of antioxidants in lung and skin tumors driven by RAS and RAF mutations, little is known about the impact of antioxidant supplementation on other cancers, including Wnt-driven tumors originating from the gut. Here, we show that supplementation with the antioxidants N-acetylcysteine (NAC) and vitamin E promotes intestinal tumor progression in the ApcMin mouse model for familial adenomatous polyposis, a hereditary form of colorectal cancer, driven by Wnt signaling. Both antioxidants increased tumor size in early neoplasias and tumor grades in more advanced lesions without any impact on tumor initiation. Importantly, NAC treatment accelerated tumor progression at plasma concentrations comparable to those obtained in human subjects after prescription doses of the drug. These results demonstrate that antioxidants play an important role in the progression of intestinal tumors, which may have implications for patients with or predisposed to colorectal cancer.